Angiogenically effective unit dose of fgf-2 and method of use

ABSTRACT

The present invention provides a unit dose composition comprising 0.2 μg/kg to 48 μg/kg of an FGF-2 of SEQ ID NO:2, or an angiogenically active fragment or mutein thereof in a pharmaceutically acceptable carrier. Also provided is a method for treating a human patient for coronary artery disease, comprising administering into one or more coronary vessels or a peripheral vein of said patient a safe and angiogenically effective dose of a recombinant FGF-2, or an angiogenically active fragment or mutein thereof. Also provided is a pharmaceutical composition comprising a therapeutically effective amount of FGF-2, alone or in combination with heparin, in a therapeutically effective carrier.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 10/184,708, filed Jun. 28, 2002 (the content of which is hereinincorporated by reference in its entirety), which is a continuationapplication of U.S. application Ser. No. 09/385,114, filed Aug. 27, 1999(the content of which is herein incorporated by reference in itsentirety), now U.S. Pat. No. 6,440,934, issued Aug. 27, 2002, whichclaims the benefit of U.S. Provisional Application Ser. No. 60/104,102,filed Oct. 13, 1998 and U.S. Provisional Application Ser. No.60/104,103, filed Oct. 13, 1998.

FIELD OF THE INVENTION

The present invention is directed to a unit dose composition forinducing cardiac angiogenesis in a human comprising a therapeuticallyeffective amount FGF-2 or an angiogenically active fragment or muteinthereof. The present invention is also directed to a method foradministering a single unit dose composition to a human to inducecardiac angiogenesis while minimizing systemic risk to the patient. Thepresent invention is useful because the disclosed unit dose composition,and method for its administration, provide an alternative to angioplastyor surgical intervention for the treatment of coronary artery disease(CAD) and further provide an adjunct for reducing post myocardialinfarct (MI) injury in humans.

BACKGROUND OF THE INVENTION

The fibroblast growth factors (FGF) are a family of at least eighteenstructurally related polypeptides (named FGF-1 to FGF-18) that arecharacterized by a high degree of affinity for proteoglycans, such asheparin. The various FGF molecules range in size from 15-23 kD, andexhibit a broad range of biological activities in normal and malignantconditions including nerve cell adhesion and differentiation [Schubertet al., J. Cell Biol. 104:635-643 (1987)]; wound healing [U.S. Pat. No.5,439,818 (Fiddes)]; as mitogens toward many mesodermal and ectodermalcell types, as trophic factors, as differentiation inducing orinhibiting factors [Clements, et al., Oncogene 8:1311-1316 (1993)]; andas an angiogenic factor [Harada, J. Clin. Invest., 94:623-630 (1994)].Thus, the FGF family is a family of pluripotent growth factors thatstimulate to varying extents fibroblasts, smooth muscle cells,epithelial cells and neuronal cells.

When FGF is released by normal tissues, such as in fetal development orwound healing, it is subject to temporal and spatial controls. However,many of the members of the FGF family are also oncogenes. Thus, in theabsence of temporal and spatial controls, they have the potential tostimulate tumor growth by providing angiogenesis.

Coronary artery disease is a progressive condition in humans wherein oneor more coronary arteries gradually become occluded through the buildupof plaque (atherosclerosis). The coronary arteries of patients havingthis disease are often treated by balloon angioplasty or the insertionof stents to prop open the partially occluded arteries. Ultimately, manyof these patients are required to undergo coronary artery bypass surgeryat great expense and risk. It would be desirable to provide suchpatients with a medicament that would enhance coronary blood flow so asto reduce the need to undergo bypass surgery.

An even more critical situation arises in humans when a patient suffersa myocardial infarction, wherein one or more coronary arteries orarterioles becomes completely occluded, such as by a clot. There is animmediate need to regain circulation to the portion of the myocardiumserved by the occluded artery or arteriole. If the lost coronarycirculation is restored within hours of the onset of the infarction,much of the damage to the myocardium that is downstream from theocclusion can be prevented. The clot-dissolving drugs, such as tissueplasminogen activator (tPA), streptokinase, and urokinase, have beenproven to be useful in this instance. However, as an adjunct to the clotdissolving drugs, it would also be desirable to also obtain collateralcirculation to the damaged or occluded myocardium by angiogenesis.

Accordingly, it is an object of the present invention to provide amedicament and a mode of administration that provides human patientswith cardiac angiogenesis during coronary artery disease and/or postacute myocardial infarction. More particularly, it is a further objectof the present invention to provide a therapeutic dose of an FGF and amode of administration to humans that provide the desired property ofcardiac angiogenesis, while minimizing adverse effects.

Many of the various FGF molecules have been isolated and administered tovarious animal models of myocardial ischemia with varying and oftentimes opposite results. According to Battler et al., “the canine modelof myocardial ischemia has been criticized because of the abundance ofnaturally occurring collateral circulation, as opposed to the porcinemodel, which ‘excels’ in its relative paucity of natural collateralcirculation and its resemblance to the human coronary circulation.”Battler et al., “Intracoronary Injection of Basic Fibroblast GrowthFactor Enhances Angiogenesis in Infarcted Swine Myocardium,” JACC,22(7): 2001-6 (December 1993) at page 2002, col. 1. However, Battler etal., who administered bovine bFGF (i.e., FGF-2) to pigs in a myocardialinfarct model, considered the varying results that are obtained from oneanimal species to another, and expressly discloses that the divergentresults “thus emphasiz[e] the caution that must be exercised inextrapolating results from different animal models.” Battler et al., atpage 2005, col. 1. Further, Battler points out that “the dosage and modeof administration of bFGF [i.e., bovine FGF-2] may have profoundimplications for the biologic effect achieved.” Battler, et al., at page2005, col. 1. Thus, it is a further object of this invention to discovera dosage and a mode of administration of a fibroblast growth factor thatwould provide for the safe and efficacious treatment of CAD and/or postMI injury in a human patient. More generally, it is an object of thepresent invention to provide a pharmaceutical composition and method forinducing angiogenesis in a human heart.

SUMMARY OF THE INVENTION

The Applicants have discovered that administering a single unit dose ofabout 0.2 μg/kg to about 48 μg/kg of rFGF-2 or an angiogenically activefragment or mutein thereof into one or more coronary vessels (IC) or aperipheral vein (IV) of a human patient in need of coronaryangiogenesis, unexpectedly provided the human patient with a rapid andtherapeutic coronary angiogenesis that resulted in an unexpectedly largeincrease (i.e., 96 and 100 seconds of increase in the mean change frombaseline for all groups at 2 and 6 months) in the treated patient'sexercise tolerance time (ETT) that persisted for an unexpectedly longduration (i.e., 6 months as of this writing). These changes shouldresult in a decreased need for standard revascularization procedures. Bythe term “coronary angiogenesis,” as used herein, is meant the formationof new blood vessels, ranging in size from capillaries to arterioleswhich act as collaterals in coronary circulation. By way of comparison,angioplasty is considered a therapeutic success if it provides anincrease in a patient's ETT of greater than 30 seconds compared to theplacebo.

Accordingly, in one aspect, the invention is directed to a unit dose ofrFGF-2 comprising a safe and therapeutically effective amount of rFGF-2or an angiogenically active fragment or mutein thereof. Typically, thesafe and therapeutically effective amount comprises about 0.2 μg/kg toabout 48 μg/kg of rFGF-2 or an angiogenically active fragment or muteinthereof, based upon ideal body weight. In other embodiments, the safeand therapeutically effective amount of the unit dose comprises 0.2μg/kg to 2.0 μg/kg, greater than 2.0 μg/kg to less than 24 μg/kg, or 24μg/kg to 48 μg/kg IC of rFGF-2 or an angiogenically active fragment ormutein thereof. In another embodiment, the safe and therapeuticallyeffective amount of the unit dose comprises 18 μg/kg to 36 μg/kg IV ofrFGF-2 or an angiogenically active fragment or mutein thereof. Expressedin absolute terms, the unit dose of the present invention comprises0.008 mg to 7.2 mg, more typically 0.3 mg to 3.5 mg, of FGF-2 or anangiogenically active fragment or mutein thereof. A suitable FGF-2 isthe rFGF-2 of SEQ ID NO:2 or an angiogenically active fragment or muteinthereof.

In another aspect, the present invention is directed to a method oftreating a human patient for CAD or to induce coronary angiogenesistherein. The method comprises administering into one or more coronaryvessels or a peripheral vein of a human patient in need of treatment forcoronary artery disease (or in need of angiogenesis) a safe andtherapeutically effective amount of a recombinant FGF-2 (rFGF-2) or anangiogenically active fragment or mutein thereof. Typically, a portionof the safe and therapeutically effective amount is administered to eachof two coronary vessels. The safe and therapeutically effective amountcomprises about 0.2 μg/kg to about 48 μg/kg of rFGF-2 or anangiogenically active fragment or mutein thereof in a pharmaceuticallyacceptable carrier. In other embodiments, the safe and therapeuticallyeffective amount comprises 0.2 μg/kg to 2 μg/kg, >2 μg/kg to <24 μg/kg,or 24 μg/kg to 48 μg/kg of rFGF-2 an angiogenically active fragment ormutein thereof in a pharmaceutically acceptable carrier. In absoluteterms, the amount of rFGF-2 or angiogenically active fragment or muteinthereof that is used in the above method comprises 0.008 mg to 7.2 mg,more typically 0.3 mg to 3.5 mg of rFGF-2 or an angiogenically activefragment or mutein thereof.

Because FGF-2 is a glycosoaminoglycan (e.g., heparin) binding proteinand the presence of a glycosoaminoglycan optimizes activity and AUC (seeFIGS. 3 and 4), the IC dosages of RFGF-2 of the present inventiontypically are administered from 0-30 minutes prior to the administrationof a glycosoaminoglycan, such as a heparin. The heparin is administeredIC or IV, typically IV. Optionally, the heparin is combined with theunit dose composition.

Because rFGF-2 releases nitric oxide, which is a potent vasodilator,aggressive fluid management prior to (proactively) and during theinfusion is critical to patient's safety. Administration of IV fluids(e.g., 500-1000 mL of normal saline) to establish a wedge pressure of 12mm Hg prior to infusion and administration of boluses of IV fluids(e.g., 200 mL normal saline) for decreases of systolic blood pressure(e.g., <90 mm Hg) associated with infusion optimized the safety ofadministration of rFGF-2 by IC or IV infusion to human patients.

Because EDTA is a potent chelator of calcium that is required for normalmyocardial contraction and cardiac conduction, minimizing theconcentration of EDTA is critical to patient's safety. A concentrationof EDTA less than 100 :g/ml in the unit dose composition optimized thesafety of administration of rFGF-2 by IC or IV infusion to humanpatients.

Because a sudden bolus of rFGF-2 is associated with profound hypotensionin animals, the rate of infusion is critical to patient's safety.Administration at 0.5 to 2 mL per minute, typically 1 mL per minute,optimized the safety of administration of rFGF-2 by IC or IV infusion tohuman patients.

The unexpected magnitude and duration of the therapeutic benefit thatwas provided to human patients in need of coronary angiogenesis by theunit dose composition and method of administration was seen as early astwo weeks after the single unit dose was administered, and persisted for6 months after the single unit dose was administered IC or IV, asdetermined by measuring art-recognized clinical endpoints such as ETT,the “Seattle Angina Questionnaire” (SAQ) and MRI of the target areas ofthe heart. In particular, when the ETT of 58 human CAD patients wasassessed by treadmill at baseline, and at 1 month, 2 months, and 6months after administration of a single unit dose of rFGF-2 by IC or IVroutes, clinical benefit was observed in some patients in all dosagegroups. See Table 1. Increases in exercise capacity appear between 1 and2 months. The mean ETT increased to greater than 60 seconds at 2 and 6months with greater benefit being seen in the higher dose group (24-48μg/kg) than in the mid (6-12 μg/kg) or low (0.33-2.0 μg/kg) dose groups.(See Table 1.) Particularly unexpected and unpredicted by animal models,were the mean increases in ETT in human patients of 93.4 and 87.5seconds that were observed at 2 and 6 months, respectively, post-dosingfor those patients administered a unit dose of rFGF-2 by IV. Evenassuming a placebo effect, the mean change from baseline for the ETTseconds still allowed an unexpectedly favorable comparison of resultswith angioplasty.

When the quality of life of 48 human CAD patients was assessed by avalidated, disease specific questionnaire, the Seattle AnginaQuestionnaire (SAQ), at baseline (i.e., prior to dosing), and at 2 and 6months after a single receiving a single unit dose of rFGF-2 of thepresent invention by IC or IV routes, the mean change from baseline forthe 5 scales measured by the SAQ increased in a clinically significantmanner for all dosage ranges whether administered IC or IV (Tables 2-6).In particular, the five scales assessed by the SAQ are exertionalcapacity, angina stability, angina frequency, treatment satisfaction,and disease perception. Relative to the baseline, the mean score forexertional capacity increased by 10.9 to 20.2 at 2 months; and by 16.5to 24.1 at 6 months. For angina stability, the mean score increased by32.1 to 46.2 at 2 months; and by 16.7 to 23.2 at 6 months. For anginafrequency, the mean score increased by 20.0 to 32.9 at 2 months; and by11.4 to 36.7 at 6 months. For treatment satisfaction, the mean scoreincreased by 8.5 to 19.8 at 2 months; and by 6.3 to 19.8 at 6 months.For disease perception, the mean score increased by 20.2 to 27.8 at 2months; and by 23.8 to 34.0 at 6 months. Generally, a change of 8 pointson any scale is considered clinically significant. Thus, the observedchanges of 8.5-46.2 are clinically significant for each of the fivescales that were assessed. Even assuming a placebo effect whereby a meanchange from baseline of 14 points is considered clinically significant,the results still provide for an unexpectedly superior effect at almostall scales that were assessed.

As part of this study, MRI was also performed on 33 human patientsdiagnosed with CAD to assess the effect of administering a single unitdose of rFGF-2 on their cardiac ejection fraction, regional myocardialfunction and perfusion (delayed arrival zone). Specifically, thepatients were administered a single unit dose of 0.33 μg/kg to 48 μg/kgIC or 18 μg/kg to 36 μg/kg IV of rFGF-2 of SEQ ID NO:2. When the 33human CAD patients were assessed by resting cardiac magnetic resonanceimaging (MRI) at baseline (i.e., prior to treatment), and 1, 2 and 6months after treatment with a single unit dose of rFGF-2 of theinvention by IC or IV routes, the patients exhibited a highlystatistically significant response to the method of treatment asobjectively measured by increased target wall thickening, target wallmotion, and target area collateral extent, and by decreased target areadelayed arrival extent. By way of summary, at 1, 2 and 6 months, thetarget wall thickening increased relative to baseline at 4.4%, 6.3% and7.7%, respectively; the target wall motion increased relative tobaseline at 2.7%, 4.4% and 6.4%, respectively; the target areacollateral extent increased relative to baseline at 8.3%, 10.9% and11.2%, respectively; and the target area delayed arrival extentdecreased relative to baseline at −10.0%, −8.3% and −10.0%,respectively.

The above data demonstrates the clinical efficacy in humans of thepresent unit dose composition of rFGF-2 or an angiogenically activefragment thereof when administered IC or IV in accordance with thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a plot of the mean rFGF-2 plasma concentration versus timeprofiles for eight different doses of rFGF-2 (SEQ ID NO:2) administeredby IC infusion in humans over a 20 minute period. The eight doses ofrFGF-2 presented in FIG. 1A are 0.33, 0.65, 2, 6, 12, 24, 36, and 48μg/kg of lean body mass (LBM).

FIG. 1B is a plot of the mean FGF-2 plasma concentration versus timeprofiles for 2 different doses of rFGF-2 (SEQ ID NO:2) administered byIV infusion in humans over a 20 minute period. The two IV doses ofrFGF-2 in FIG. 1B are 18 and 36 μg/kg. The mean concentration-timeprofile following IC administration of 36 μg/kg rFGF-2 is included forcomparison.

FIG. 2 is a plot of mean FGF-2 area under the curve (AUC) in pg*min/mlcorresponding to FIGS. 1A and 1B. This plot shows the dose linearity ofsystemic rFGF-2 exposure following IC or IV infusion. The systemicexposure for the IC route is similar to that observed following IVadministration.

FIG. 3 is a plot of individual human patient FGF-2 plasma clearance (CL)values as a function of the time of heparin administration in “minutesprior to rFGF-2 infusion” and shows the influence of timing of heparinadministration on rFGF-2 plasma clearance (CL).

FIG. 4 is a plot individual human patient FGF-2 dose normalized areaunder curves (AUCs) as a function of the time of heparin administrationin “minutes prior to rFGF-2 infusion” and shows the influence of timingof heparin administration on FGF-2 AUC.

DETAILED DESCRIPTION OF THE INVENTION

The Applicants have discovered that a single dose of rFGF-2 or anangiogenically active fragment or mutein thereof, when administered in asafe and therapeutically effective amount into one or more coronaryvessels or into a peripheral vein of a human patient diagnosed with CADprovides the patient with a safe and therapeutically efficacioustreatment for the patient's coronary artery disease that lasts at least4 to 6 months, more typically at least six months, before a furthertreatment is needed. This duration of the effect and the magnitude ofthe improvements in ETT, SAQ and MRI were unexpected for a single unitdose of medicament.

By the phrase “therapeutically effective amount” or “safe andtherapeutically effective amount” as used herein in relation to rFGF-2is meant an amount of rFGF-2 or an angiogenically active fragment ormutein thereof that when administered in accordance with this invention,is free from major complications that cannot be medically managed, andthat provides for objective cardiac improvement in patients havingsymptoms of CAD despite optimum medical management. Thus, acutehypotension that can be managed by administration of fluids, isconsidered “safe” for the purpose of this invention. Typically, the safeand therapeutically effective amount of rFGF-2 comprises about 0.2 μg/kgto about 48 μg/kg of rFGF-2 or an angiogenically active fragment ormutein thereof. A suitable FGF-2 for use in the present invention is therFGF-2 of SEQ ID NO:2 or an angiogenically active fragment or muteinthereof.

Accordingly, the present invention has multiple aspects. In its firstaspect, the present invention is directed to a unit dose composition forinducing angiogenesis in a human patient, the unit dose comprising atherapeutically effective (i.e., an angiogenically effective) amount ofrFGF-2 or an angiogenically active fragment or mutein thereof, theamount comprising about 0.2 μg/kg to about 48 μg/kg of rFGF-2 or anangiogenically active fragment or mutein thereof.

By the term “unit dose composition” as used herein is meant acomposition that when administered to a human patient in accordance withthe method of the present invention provides a typical human patient inneed of angiogenesis with an angiogenic effect of significant efficacyso as not to require retreatment for at least 4-6 months, typically 6months. The unit dose composition of the present invention is typicallyprovided in combination with one or more pharmaceutically acceptableexcipients or carriers. In other embodiments of the unit dosecomposition, a safe and therapeutically effective amount comprises about0.2 μg/kg to about 2 μg/kg, about 2 μg/kg to about 24 μg/kg, or about 24μg/kg to about 48 μg/kg of rFGF-2 or an angiogenically active fragmentor mutein thereof.

It is convenient to define the unit dose composition of the presentinvention in more absolute terms that are not dependent upon the weightof the patient to be treated. When so defined, the unit dose compositioncomprises from 0.008 mg to 7.2 mg of rFGF-2 or an angiogenically activefragment or mutein thereof. In this embodiment, the unit dosecomposition contains a sufficient amount of FGF-2 to accommodate dosingany one of the majority of human CAD patients, ranging from the smallestpatient (e.g., 40 kg) at the lowest dosage (about 0.2 μg/kg) through thelarger patients (e.g., 150 kg) at the highest dosage (about 48 μg/kg).More typically, the unit dose comprises 0.3 mg to 3.5 mg of rFGF-2 or anangiogenically active fragment or mutein thereof. The unit dosecomposition is typically provided in solution or lyophilized formcontaining the above referenced amount of rFGF-2 and an effective amountof one or more pharmaceutically acceptable buffers, stabilizers and/orother excipients as later described herein.

The active agent in the Applicants' above described unit dosecomposition is a recombinant FGF-2 or an angiogenically active fragmentor mutein thereof. Methods for making recombinant FGF-2 are well-knownin the art. The recombinant FGF-2 of SEQ ID NO:2 is made as described inU.S. Pat. No. 5,155,214, entitled “Basic Fibroblast Growth Factor,”which issued on Oct. 13, 1992, and which is expressly incorporatedherein by reference in its entirety. Moreover, all other referencescited herein, whether occurring before or after this sentence, areexpressly incorporated herein by reference in their entirety. Asdisclosed in the '214 patent, a DNA of SEQ ID NO:1, which encodes a bFGF(hereinafter “FGF-2”) of SEQ ID NO:2, is inserted into a cloning vector,such as pBR322, pMB9, Col E1, pCR1, RP4 or λ-phage, and the cloningvector is used to transform either a eukaryotic or prokaryotic cell,wherein the transformed cell expresses the FGF-2. In one embodiment, thehost cell is a yeast cell, such as Saccharomyces cerevisiae. Theresulting full length FGF-2 that is expressed has 146 amino acids inaccordance with SEQ ID NO:2. Although the FGF-2 of SEQ ID NO:2 has fourcysteines, i.e., at residue positions 25, 69, 87 and 92, there are nointernal disulfide linkages. ['214 at col. 6, lines 59-61.] However, inthe event that cross-linking occurred under oxidative conditions, itwould likely occur between the residues at positions 25 and 69.

The FGF-2 of SEQ ID NO:2, which has 146 amino acid residues, differsfrom naturally occurring human FGF-2 by only two amino acid residue. Inparticular, the amino acids at residue positions 112 and 128 of theFGF-2 of SEQ ID NO:2 are Ser and Pro, respectively, whereas in humanFGF-2, they are Thr and Ser, respectively. In nature, bovine FGF-2, likethe corresponding human FGF-2 is initially synthesized in vivo as apolypeptide having 155 amino acid residues. Abraham et al. “Human BasicFibroblast Growth Factor: Nucleotide Sequence and Genomic Organization,”EMBO J., 5(10):2523-2528 (1986). When the FGF-2 of SEQ ID NO:2 iscompared to the full length 155 residue bovine FGF-2 of Abraham, theFGF-2 of SEQ ID NO:2 lacks the first nine amino acid residues, Met AlaAla Gly Ser Ile Thr Thr Leu (SEQ ID NO:3), at the N-terminus of thecorresponding full length molecule. The recombinant FGF-2 employed inthe present compositions and method was purified to pharmaceuticalquality (98% or greater purity) using the techniques described in detailin U.S. Pat. No. 4,956,455, entitled “Bovine Fibroblast Growth Factor”which issued on Sep. 11, 1990 and which is incorporated herein byreference in its entirety. In particular, the first two steps employedin the purification of the recombinant FGF-2 of Applicants' unit dosecomposition are “conventional ion-exchange and reverse phase HPLCpurification steps as described previously.” [U.S. Pat. No. 4,956,455,citing to Bolen et al., PNAS USA 81:5364-5368 (1984).] The third step,which the '455 patent refers to as the “key purification step” ['455 atcol. 7, lines 5-6], is heparin-SEPHAROSE® affinity chromatography,wherein the strong heparin binding affinity of the FGF-2 is utilized toachieve several thousand-fold purification when eluting at approximately1.4M and 1.95M NaCl ['455 at col. 9, lines 20-25]. Polypeptidehomogeneity was confirmed by reverse-phase high pressure liquidchromatography (RP-HPLC). Buffer exchange was achieved by SEPHADEX®G-25(M) gel filtration chromatography.

In addition to the 146 residue rFGF-2 of SEQ ID NO:2, the active agentin the unit dose of the present invention also comprises an“angiogenically active fragment” of FGF-2. By the term “angiogenicallyactive fragment” of FGF-2 is meant a fragment of FGF-2 that has about80% of the 146 residues of SEQ ID NO:2 and that retains at least 50%,preferably at least 80%, of the angiogenic activity of the FGF-2 of SEQID NO:2.

To be angiogenically active, the FGF-2 fragment should have two cellbinding sites and at least one of the two heparin binding sites. The twoputative cell binding sites of the analogous human FGF-2 occur atresidue positions 36-39 and 77-81 thereof. See Yoshida, et al., “GenomicSequence of hst, a Transforming Gene Encoding a Protein Homologous toFibroblast Growth Factors and the int-2-Encoded Protein,” PNAS USA,84:7305-7309 (October 1987) at FIG. 3. The two putative heparin bindingsites of hFGF-2 occur at residue positions 18-22 and 107-111 thereof.See Yoshida (1987) at FIG. 3. Given the greater than 98% similaritybetween the amino acid sequences for naturally occurring human FGF-2(hFGF-2) and rFGF-2 (SEQ ID NO:2), it is expected that the two cellbinding sites for rFGF-2 (SEQ ID NO:2) are also at residue positions36-39 and 77-81 thereof, and that the two heparin binding sites are atresidue positions 18-22 and 107-111 thereof. Consistent with the above,it is well known in the art that N-terminal truncations of the FGF-2 ofSEQ ID NO:2 do not eliminate its activity in cows. In particular, theart discloses several naturally occurring and biologically activefragments of the FGF-2 that have N-terminal truncations relative to theFGF-2 of SEQ ID NO:2. An active and truncated bFGF-2 having residues12-146 of SEQ ID NO:2 was found in bovine liver and another active andtruncated bFGF-2, having residues 16-146 of SEQ ID NO:2 was found in thebovine kidney, adrenal glands and testes. [See U.S. Pat. No. 5,155,214at col. 6, lines 41-46, citing to Ueno, et al., Biochem and Biophys Res.Comm., 138:580-588 (1986).] Likewise, other fragments of the bFGF-2 ofSEQ ID NO:2 that are known to have FGF activity are FGF-2 (24-120)-OHand FGF-2 (30-110)-NH₂. [U.S. Pat. No. 5,155,214 at col. 6, lines48-52.] These latter fragments retain both of the cell binding portionsof FGF-2 (SEQ ID NO:2) and one of the heparin binding segments (residues107-111). Accordingly, the angiogenically active fragments of FGF-2typically encompass those terminally truncated fragments of FGF-2 thathave at least residues that correspond to residues 30-110 of FGF-2 ofSEQ ID NO:2; more typically, at least residues that correspond toresidues 18-146 of FGF-2 of SEQ ID NO:2.

The unit dose of the present invention also comprises an “angiogenicallyactive . . . mutein” of the rFGF-2 of SEQ ID NO:2. By the term“angiogenically active . . . mutein” as used herein, is meant anisolated and purified recombinant protein or polypeptide that has 65%sequence identity (homology) to any naturally occurring FGF-2, asdetermined by the Smith-Waterman homology search algorithm (Meth. Mol.Biol. 70:173-187 (1997)) as implemented in MSPRCH program (OxfordMolecular) using an affine gap search with the following searchparameters: gap open penalty of 12, and gap extension penalty of 1, andthat retains at least 50%, preferably at least 80%, of the angiogenicactivity of the naturally occurring FGF-2 with which it has said atleast 65% sequence identity. Preferably, the angiogenically activemutein has at least 75%, more preferably at least 85%, and mostpreferably, at least 90% sequence identity to the naturally occurringFGF-2. Other well-known and routinely used homology/identity scanningalgorithm programs include Pearson and Lipman, PNAS USA, 85:2444-2448(1988); Lipman and Pearson, Science, 222:1435 (1985); Devereaux et al.,Nuc. Acids Res., 12:387-395 (1984); or the BLASTP, BLASTN or BLASTXalgorithms of Altschul, et al., Mol. Biol., 215:403-410 (1990).Computerized programs using these algorithms are also available andinclude, but are not limited to: GAP, BESTFIT, BLAST, FASTA and TFASTA,which are commercially available from the Genetics Computing Group (GCG)package, Version 8, Madison Wis., USA; and CLUSTAL in the PC/Geneprogram by Intellegenetics, Mountain View Calif. Preferably, thepercentage of sequence identity is determined by using the defaultparameters determined by the program.

The phrase “sequence identity,” as used herein, is intended to refer tothe percentage of the same amino acids that are found similarlypositioned within the mutein sequence when a specified, contiguoussegment of the amino acid sequence of the mutein is aligned and comparedto the amino acid sequence of the naturally occurring FGF-2.

When considering the percentage of amino acid sequence identity in themutein, some amino acid residue positions may differ from the referenceprotein as a result of conservative amino acid substitutions, which donot affect the properties of the protein or protein function. In theseinstances, the percentage of sequence identity may be adjusted upwardsto account for the similarity in conservatively substituted amino acids.Such adjustments are well-known in the art. See, e.g., Meyers andMiller, “Computer Applic. Bio. Sci., 4:11-17 (1988).

To prepare an “angiogenically active mutein” of an angiogenic agent ofthe present invention, one uses standard techniques for site directedmutagenesis, as known in the art and/or as taught in Gilman, et al.,Gene, 8:81 (1979) or Roberts, et al., Nature, 328:731 (1987). Using oneof the site directed mutagenesis techniques, one or more point mutationsare introduced into the cDNA sequence of SEQ ID NO: 1 to introduce oneor more amino acid substitutions or an internal deletion. Conservativeamino acid substitutions are those that preserve the general charge,hydrophobicity/hydrophilicity, and/or steric bulk of the amino acidbeing substituted. By way of example, substitutions between thefollowing groups are conservative: Gly/Ala, Val/Ile/Leu, Lys/Arg,Asn/Gln, Glu/Asp, Ser/Cys/Thr, and Phe/Trp/Tyr. Significant (up to 35%)variation from the sequence of the naturally occurring angiogenic FGF-2is permitted as long as the resulting protein or polypeptide retainsangiogenic activity within the limits specified above.

Cysteine-depleted muteins are muteins within the scope of the presentinvention. These muteins are constructed using site directed mutagenesisas described above, or according to the method described in U.S. Pat.No. 4,959,314 (“the '314 patent”), entitled “Cysteine-Depleted Muteinsof Biologically Active Proteins.” The '314 patent discloses how todetermine biological activity and the effect of the substitution.Cysteine substitution is particularly useful in proteins having two ormore cysteines that are not involved in disulfide formation. Suitablesubstitutions include the substitution of serine for one or both of thecysteines at residue positions 87 and 92, which are not involved indisulfide formation. Preferably, substitutions are introduced at theFGF-2 N-terminus, which is not associated with angiogenic activity.However, as discussed above, conservative substitutions are suitable forintroduction throughout the molecule.

The unit dose composition of the present invention comprises a safe andan angiogenically effective dose of rFGF-2 or an angiogenically activefragment or mutein thereof, and a pharmaceutically acceptable carrier.Typically, the safe and angiogenically effective dose of thepharmaceutical composition of the present invention is in a form and asize suitable for administration to a human patient and comprises (i)0.2 μg/kg to 48 μg/kg of rFGF-2 or an angiogenically active fragment ormutein thereof, (ii) and a pharmaceutically acceptable carrier. In otherembodiments, the safe and angiogenically effective dose comprises 0.2μg/kg to 2 μg/kg, >2 μg/kg to <24 μg/kg or 24 μg/kg to 48 μg/kg of FGF-2or an angiogenically active fragment or mutein thereof, and apharmaceutically acceptable carrier. Expressed in absolute terms for themajority of human CAD patients, the unit dose of the present inventioncomprises 0.008 mg to 7.2 mg, more typically 0.3 mg to 3.5 mg, of theFGF-2 or an angiogenically active fragment or mutein thereof.

The second recited component of the unit dose composition of the presentinvention is a “pharmaceutically acceptable carrier.” By the term“pharmaceutically acceptable carrier” as used herein is meant any of thecarriers or diluents that are well-known in the art for thestabilization and/or administration of a proteinaceous medicament thatdoes not itself induce the production of antibodies harmful to thepatient receiving the composition, and which may be administered withoutundue toxicity. The choice of the pharmaceutically acceptable carrierand its subsequent processing enables the unit dose composition of thepresent invention to be provided in either liquid or solid form.

When the unit dose composition is in liquid form, the pharmaceuticallyacceptable carrier comprises a stable carrier or diluent suitable forintravenous (“IV”) or intracoronary (“IC”) injection or infusion.Suitable carriers or diluents for injectable or infusible solutions arenontoxic to a human recipient at the dosages and concentrationsemployed, and include sterile water, sugar solutions, saline solutions,protein solutions or combinations thereof.

Typically, the pharmaceutically acceptable carrier includes a buffer andone or more stabilizers, reducing agents, anti-oxidants and/oranti-oxidant chelating agents. The use of buffers, stabilizers, reducingagents, anti-oxidants and chelating agents in the preparation of proteinbased compositions, particularly pharmaceutical compositions, iswell-known in the art. See, Wang et al., “Review of Excipients and pHsfor Parenteral Products Used in the United States,” J. Parent. DrugAssn., 34(6):452-462 (1980); Wang et al., “Parenteral Formulations ofProteins and Peptides: Stability and Stabilizers,” J. Parent. Sci. andTech., 42:S4-S26 (Supplement 1988); Lachman, et al., “Antioxidants andChelating Agents as Stabilizers in Liquid Dosage Forms-Part 1,” Drug andCosmetic Industry, 102(1): 36-38, 40 and 146-148 (1968); Akers, M. J.,“Antioxidants in Pharmaceutical Products,” J. Parent. Sci. and Tech.,36(5):222-228 (1988); and Methods in Enzymology, Vol. XXV, Colowick andKaplan Eds., “Reduction of Disulfide Bonds in Proteins withDithiothreitol,” by Konigsberg, pages 185-188. Suitable buffers includeacetate, adipate, benzoate, citrate, lactate, maleate, phosphate,tartarate and the salts of various amino acids. See Wang (1980) at page455. Suitable stabilizers include carbohydrates such as threlose orglycerol. Suitable reducing agents, which maintain the reduction ofreduced cysteines, include dithiothreitol (DTT also known as Cleland'sreagent) or dithioerythritol at 0.01% to 0.1% wt/wt; acetylcysteine orcysteine at 0.1% to 0.5% (pH 2-3); and thioglycerol at 0.1% to 0.5% (pH3.5 to 7.0) and glutathione. See Akers (1988) at pages 225 to 226.Suitable antioxidants include sodium bisulfite, sodium sulfite, sodiummetabisulfite, sodium thiosulfate, sodium formaldehyde sulfoxylate, andascorbic acid. See Akers (1988) at pages 225. Suitable chelating agents,which chelate trace metals to prevent the trace metal catalyzedoxidation of reduced cysteines, include citrate, tartarate,ethylenediaminetetraacetic acid (EDTA) in its disodium, tetrasodium, andcalcium disodium salts, and diethylenetriamine pentaacetic acid (DTPA).See e.g., Wang (1980) at pages 457-458 and 460-461, and Akers (1988) atpages 224-227. Suitable sugars include glycerol, trehalose, glucose,galactose and mannitol, sorbitol. A suitable protein is human serumalbumin.

In liquid form, a typical unit dose composition of the present inventioncomprises from about 0.001 mg to 8 mg, more typically 0.03 to 5 mgrFGF-2 or an angiogenically active fragment or mutein thereof, dissolveda pharmaceutically acceptable carrier. A suitable pharmaceuticallyacceptable carrier comprises 10 mM thioglycerol, 135 mM NaCl, 10 mMsodium citrate, and 1 mM EDTA, pH 5. A suitable diluent or flushingagent for the above-described unit dose composition is any of theabove-described carriers. Typically, the diluent is the carriersolution. rFGF-2 or an angiogenically active fragment or mutein thereofis unstable for long periods of time in liquid form. To maximizestability and shelf life of the liquid form, the unit dose compositionshould be stored frozen at −60° C. When thawed, the solution is stablefor 6 months at refrigerated conditions. A typical unit dose wouldcomprise about 1-40 ml, more typically 10-40 ml, of the above-describedcomposition having 0.008-7.2 mg of rFGF-2 or an angiogenically activefragment or mutein dissolved therein. A suitable rFGF-2 for use in theunit dose is the rFGF-2 of SEQ ID NO:2 or an angiogenically activefragment or mutein thereof.

In another embodiment, the unit dose composition is provided inlyophilized (freeze-dried) form. In this form, the unit dose of rFGF-2is capable of being stored at refrigerated temperatures forsubstantially longer than 6 months without loss of therapeuticeffectiveness. Lyophilization is accomplished by the rapid freeze drying(i.e., removing water) under reduced pressure of a plurality of vials,each containing the above described liquid form of the unit dose of therFGF-2 of the present invention therein. Lyophilizers, which perform theabove described lyophilization, are commercially available and readilyoperable by those skilled in the art. The resulting lyophilized unitdose composition, in lyophilized cake form, is formulated to containwithin the resulting lyophilized cake one or more of the buffers,stabilizers, anti-oxidants, reducing agents, salts and/or sugarsdescribed above for the corresponding liquid formulation. A lyophilizedunit dose composition containing all such other components need only bereconstituted to a known volume or concentration with sterile aqueousdiluent such as sterile water, a sterile sugar solution, or a sterilesaline solution. Alternatively, it could be reconstituted with a sterilebuffer solution as described above, but lacking a chelating agent, suchas EDTA. As a lyophilized cake, the unit dose composition is stable from6 months to two years at refrigerated temperatures. Thus, storage of theunit dose composition in lyophilized form is readily accommodated usingconventional refrigeration equipment.

Because the unit dose composition of the present invention isadministered via a cardiac catheter or other injection device, which hasdead space, it is convenient to formulate the vial containing the unitdose composition so that it contains about 10-50% more of the rFGF-2 orangiogenically active fragment or mutein thereof than is to beadministered to the patient. For example, when the unit dose of therFGF-2 to be administered is 7.2 mg, the vial is optionally formulatedto contain up to 50% extra (e.g., a total of about 10.8 mg) of rFGF-2 orangiogenically active fragment or mutein thereof. The extra solution issuitable for filling the dead space in the delivery equipment. In analternative embodiment that does not allow for dead space, thepharmaceutical composition is loaded in the cardiac catheter in front ofa pharmaceutically acceptable buffer, diluent or carrier, which is thenused to deliver the appropriate amount of the one or more dosages to theone or more sites in the myocardium that are in need of angiogenesis.

As discussed above, the pharmaceutically acceptable carrier for theabove described unit dose composition comprises a buffer and one or morestabilizers, reducing agents, anti-oxidants and/or anti-oxidantchelating agents. It is also within the scope of the present inventionthat the unit dose composition contain an amount of a glycosoaminoglycan(also known as a “proteoglycan” or a “mucopolysaccharide”), such asheparin, that is effective to bind to the FGF-2 and to the endothelialcell receptors so as to enhance the angiogenic effectiveness of theFGF-2 or angiogenically active fragment or mutein thereof. The amount ofheparin that is administered is about 10-80 U per kg of patient weight(U/kg), typically about 40 U/kg. Expressed in absolute terms, the totalamount of heparin administered to any one patient does not exceed 5,000U. Thus, upon reconstitution, the unit dose composition of the presentinvention would not only contain an angiogenically effective amount ofrFGF-2 or an angiogenically active fragment or mutein thereof, it wouldalso contain from about 10-80 U/kg of heparin, typically about 40 U/kg.The typical volume of diluent is from about 1 to 40 ml. While largervolumes of diluent could be used, such larger volumes would typicallyresult in longer administration times. Depending upon the weight of thepatient in kg, a single dose comprising from 0.2 μg/kg to 48 μg/kg ofthe rFGF-2 or an angiogenically active fragment or mutein thereof iswithdrawn from the vial as reconstituted product for administration tothe patient. Thus, an average 70 kg man that is being dosed at 24 μg/kgwould have a sufficient volume of the reconstituted product withdrawnfrom the vial to receive an IC infusion of (70 kg×30 μg/kg) 2100 μg(i.e., 2.1 mg).

In its second aspect, the present invention is directed to a method fortreating a human patient for CAD or MI, using the above described unitdose composition. In particular, in one embodiment, the presentinvention is directed to a method for treating a human patient forcoronary artery disease, comprising administering a safe andtherapeutically effective amount of a recombinant FGF-2 or anangiogenically active fragment or mutein thereof to one or more,typically two, patent coronary vessels or a peripheral vein of a humanpatient in need of treatment for coronary artery disease. The humanpatient in need of treatment for coronary artery disease is typically ahuman patient with coronary artery disease who remains symptomatic withangina despite optional medical management. A preferred coronary vesselis a coronary artery, although grafted saphenous veins and graftedinternal mammary arteries, as provided by coronary angioplasty, are alsosuitable. Suitable peripheral veins for administering the unit dosecomposition include those peripheral veins found throughout the humanbody that are routinely used by treating physicians and nurses foradministration of fluids and medicaments. Examples of such veins includethe cephalic, the median cubital, and the basilic of the arm.

When administered as an intracoronary (IC) infusion, the unit dose ofrFGF-2 or angiogenic fragment or mutein thereof is typicallyadministered within an hour, more typically over a period of about 20minutes into one or more (typically, two) patent coronary vessels. Whenadministered over a twenty minute period, the unit dose composition istypically administered at a rate of 0.5 to 2.0 ml/minute, more typicallyat about 1 ml/minute. The coronary vessels can be native vessels orgrafts, so long as they are not occluded. The volume of the unit dose ofrFGF-2 or angiogenic fragment or mutein thereof is typically 10-40 ml;more typically 20 ml. The length of time for infusion of the unit doseis not critical and can be shortened or lengthened depending on the rateand volume of infusion.

When administered as an intravenous (IV) infusion, the unit dose ofrFGF-2 or angiogenic fragment or mutein thereof is administeredtypically within an hour, more typically over a 20 minute period, into aperipheral vein using a conventional IV setup. When administered over atwenty minute period, the unit dose composition is typicallyadministered at a rate of 1 ml/minute.

In the phase I clinical trial of the above described method for treatingCAD, a single unit dose composition was administered IC or IV to humanpatients having CAD who remained symptomatic with angina despiteoptional medical management. Because the method of the present inventioninduces angiogenesis, the method of the present invention providestreatment of the underlying condition in CAD or MI and not merelytransitory relief from the symptoms, such as provided by nitrates.Typically, the safe and therapeutically effective amount of the methodof the present invention comprises 0.2 μg/kg to 48 μg/kg of rFGF-2 or anangiogenically active fragment or mutein thereof in a pharmaceuticallyacceptable carrier. In other embodiments, the safe and therapeuticallyeffective amount comprises 0.2 μg/kg to 2 μg/kg, >2 μg/kg to <24 μg/kg,or 24 μg/kg to 48 μg/kg of rFGF-2 or an angiogenically active fragmentor mutein thereof in a pharmaceutically acceptable carrier. In absoluteterms, the safe and therapeutically effective amount is about 0.008 mgto about 7.2 mg of rFGF-2 or an angiogenically active fragment or muteinthereof; more typically, 0.3 mg to 3.5 mg of rFGF-2 or an angiogenicallyactive fragment or mutein thereof. A suitable rFGF-2 is the rFGF-2 ofSEQ ID NO:2 or an angiogenically active fragment or mutein thereof.

In another aspect, the present invention is also directed to a methodfor inducing angiogenesis in a heart of a human patient comprising,administering a single unit dose composition of a recombinant FGF-2 oran angiogenically active fragment or mutein thereof to one or morecoronary vessels or to a peripheral vein in a human patient in need ofcoronary angiogenesis, said unit dose composition comprising from about0.008 mg to 7.2 mg of recombinant rFGF-2 or an angiogenically activefragment or mutein thereof in a pharmaceutically acceptable carrier.More typically, the unit dose composition comprises about 0.3-3.5 mgrFGF-2 or an angiogenically active fragment or mutein thereof in apharmaceutically acceptable carrier. As described above, a single unitdose composition containing a therapeutically effective amount of anrFGF-2 or an angiogenically fragment or mutein thereof is administeredto at least one coronary vessel of a human patient in need ofangiogenesis, using standard cardiac catheterization techniques alreadyknown and used in the art for the intracoronary administration ofmedicaments, e.g., thrombolytics, streptokinase, or radio-opaque dyes ormagnetic particles used to visualize the coronary arteries. By way ofexample, a coronary catheter is inserted into an artery (e.g., femoralor subclavian) of the patient in need of treatment and the catheter ispushed forward, with visualization, until it is positioned in theappropriate coronary vessel of the patient to be treated. Using standardprecautions for maintaining a clear line, the pharmaceutical compositionin solution form is administered by infusing the unit dose substantiallycontinuously over a period of 10 to 30 minutes. Although thepharmaceutical composition of the invention could be administered over alonger period of time, the Applicants perceive no benefit and apotentially increased risk of thrombosis in doing so. Typically, aportion (e.g., one half) of the unit dose is administered in a firstcoronary vessel. Then, the catheter is repositioned into a secondsecondary coronary vessel and the remainder of the unit dose isadministered with flushing of the catheter. Using the above-describedrepositioning procedure, portions of the unit dose may be administeredto a plurality of coronary vessels until the entire unit dose has beenadministered. After administration, the catheter is withdrawn usingconventional art known protocols. In the phase I clinical trialsdescribed herein, therapeutic benefit was reported by patients as earlyas two weeks following the IC rFGF-2 administration of a single unitdose. Clinically significant improvement was demonstrable by objectivecriterion (ETT and/or SAQ) as early as 30 days following IC or IVadministration of a single unit dose of the present invention, and wasmaintained for six months following dosing. In certain patients withprogressive CAD disease, it may be necessary or appropriate toadminister additional unit doses of rFGF-2 at six or 12 month intervalsafter the initial unit dose, to overcome the progression of the CADduring that interim period. In some patients with very progressive CAD,unit doses of present invention would be readministered at 4 monthintervals. In any instance, the treating physician would be able todetermine the time, if any, for readministration based upon routineassessment of the clinical symptoms of the patient.

One of the benefits of the method of the present invention is cardiacangiogenesis. Accordingly, in another aspect, the present invention isdirected to a method for inducing angiogenesis in a heart of a humanpatient, comprising administering into one or more coronary vessels (IC)or into a peripheral vein (IV) of a human patient in need of coronaryangiogenesis, a single unit dose composition comprising anangiogenically effective amount of rFGF-2 or an angiogenically activefragment or mutein thereof in a pharmaceutically acceptable carrier. Inthe above method, the angiogenically effective amount comprises about0.2 μg/kg to about 48 μg/kg (or in absolute terms about 0.008 mg toabout 7.2 mg) of a recombinant FGF-2 or an angiogenically activefragment or mutein thereof. More typically, the angiogenically effectiveamount comprises about 0.3 mg to 3.5 mg of a recombinant FGF-2 or anangiogenically active fragment or mutein thereof. A suitable rFGF-2 foruse in the above-identified method is the rFGF-2 of SEQ ID NO:2 or anangiogenically active fragment thereof. In one embodiment of the abovemethod, the unit dose composition is administered IC to patent coronaryvessels or IV to a peripheral vein. In another embodiment, the unit dosecomposition is administered with heparin as described herein.

The above described method for providing coronary angiogenesis is alsobeneficial in human patients that have undergone a myocardial infarction(MI) in one or more coronary arteries. Accordingly, in another aspect,the present invention is also directed to a method for treating a humanpatient for an MI comprising, administering into one or more coronaryvessels or into a peripheral vein of said human patient, a single unitdose composition comprising a therapeutically effective amount of rFGF-2or an angiogenically active fragment or mutein thereof. In the abovemethod, the unit dose composition typically comprises about 0.2 μg/kg toabout 48 μg/kg (or in absolute terms about 0.008 mg to about 7.2 mg) ofa recombinant FGF-2 or an angiogenically active fragment or muteinthereof in a pharmaceutically acceptable carrier. A suitable rFGF-2 foruse in the above-identified method is the rFGF-2 of SEQ ID NO:2 or anangiogenically active fragment thereof.

In the event of unstable angina or acute myocardial infarction,requiring angioplasty, the same doses of rFGF-2 or angiogenic fragmentor mutein thereof that are disclosed herein would also be useful as anadjunct therapy in treating those conditions. Accordingly, in anotheraspect, the present invention is directed to an improved method fortreating a patient for unstable angina or acute myocardial infarction,requiring angioplasty, the method comprising providing angioplasty tothe patient in need of treatment; the improvement comprisingadministering into one or more coronary vessels or into a peripheralvein of said human patient, a single unit dose composition comprising atherapeutically effective amount of rFGF-2 or an angiogenically activefragment or mutein thereof. In the above method, the unit dosecomposition comprises about 0.2 μg/kg to about 48 μg/kg (or in absoluteterms about 0.008 mg to about 7.2 mg) of a recombinant FGF-2 or anangiogenically active fragment or mutein thereof in a pharmaceuticallyacceptable carrier. A suitable rFGF-2 for use in the above-identifiedmethod is the rFGF-2 of SEQ ID NO:2 or an angiogenically active fragmentthereof.

In any of the above-described methods of the present invention, therFGF-2 or the angiogenically active fragment or mutein thereof isassociated with release of nitric oxide, a recognized smooth muscledilator, which upon administration to the patient causes a sudden dropin the patient's blood pressure. Accordingly, in the methods of thepresent invention, it is preferable to hydrate the patient with IVfluids prior to administering the unit dose of the present invention.Moreover, for safety and tolerability of the unit dose, aggressive fluidmanagement during and after rFGF-2 administration is also preferred.Finally, it is also within the scope of the above described methods toinclude the step of administering an effective amount of aglycosoaminoglycan (also known as a “proteoglycan” or a“mucopolysaccharide”), such as heparin from 0-30 minutes prior toadministering the unit dose composition of the present invention.Typically, the effective amount of glycosaminoglycan (such as heparin)that is administered is about 10-80 U/kg, more typically, about 40 U/kg.However, the total amount of heparin administered to any one patientimmediately prior to dosing generally does not exceed 5,000 U.

Because EDTA is a potent chelator of calcium which is required fornormal myocardial contraction and cardiac conduction, minimizing theconcentration of EDTA is critical to patient's safety. A concentrationof EDTA less than 100 μg/ml optimized the safety of administration ofrFGF-2 by IC or IV infusion to human patients.

Because a sudden bolus of rFGF-2 is associated with profound hypotensionin animals, the rate of infusion is critical to patient's safety.Administration at 0.5 to 2 mL per minute, typically 1 mL per minute,optimized the safety of administration of rFGF-2 by IC or IV infusion tohuman patients.

A Phase I clinical trial directed to treating human patients for CAD byadministering a single unit dose composition of the present inventionwas conducted and is described in Examples 1-3 herein. In that trial,sixty-six (66) human patients diagnosed with CAD, who satisfied thecriteria of Example 2 herein, received a single unit dose of rFGF-2 inaccordance with the method of the present invention. Specifically,fifty-two human patients were administered a unit dose of 0.33 μg/kg to48 μg/kg of rFGF-2 by IC infusion over about a 20 minute period.Fourteen human patients were administered a unit dose of either 18 μg/kgor 36 μg/kg of rFGF-2 by IV infusion over about a 20 minute period. The66 treated patients were then assessed relative to baseline (i.e., priorto treatment with the single unit dose), and again at 1 month, 2 monthsand 6 months after treatment with the single unit dose, using three setsof art-recognized assessment criteria: 1) changes in their exercisetolerance time (ETT); 2) the Seattle Angina Questionnaire, whichprovides an assessment based upon a mixed combination of objective andsubjective criteria; and 3) the measurement of physical changes in theheart as assessed by MRI.

For ETT of the 66 patients of the Phase I clinical trial of Examples 1-3was measured at baseline, and at 1 month, 2 months and 6 months afterdosing (with a single unit dose composition of the invention) using aBruce treadmill protocol. Subjects were excluded from the analysis ifthe treadmill protocol was not the same as used at baseline. Therefore,the number of subjects varied over time. In addition, any patients whohad emergency revascularization were excluded from the analysis. A dosewas considered effective if the mean change in ETT from baselineincreased by greater than 60 seconds. The results of the ETT assessmentare provided in Table 1.

TABLE 1 Exercise Tolerance Time (ETT) - Change from Baseline Change fromChange from Change from FGF-2 Baseline Baseline Baseline Dose Group atOne Month at Two Months at Six Months 0.33 to 2.0 μg/kg IC N = 8  N = 6 N = 5  (N = 16) 45.1 sec 130.0 sec* 60.8 sec (low) (−105 to 180)  (19 to240) (−45 to 210) 6.0 and 12 μg/kg IC N = 2  N = 4  N = 2  (N = 8) −24.0sec  −2.5 sec  6.5 sec (mid) (−48 to 0)  (−90 to 120) (−0 to 13) 24.0 to48.0 μg/kg IC N = 18 N = 21 N = 16 (N = 28) 51.9 sec 107.9 sec* 133.1sec* (high) (−188 to 399) (−30 to 385) (−195 to 386)  18.0 & 36.0 μg/kgIV N = 12 N = 12 N = 12 (N = 14) 45.1 sec  93.4 sec*  87.5 sec* (−75 to237)   (0 to 285) (−60 to 285) ALL GROUPS N = 40 N = 43 N = 35 (N = 66)45.0 sec 96.0 sec 100.0 sec  N = number of subjects; mean; (range inseconds); *= p < 0.05

Referring to Table 1, the mean change from baseline at one month wasless than 60 seconds for all dose groups. However, the percentage ofpatients stopping their treadmill test because of angina decreased inall groups over time. At 2 months and 6 months after dosing, the meanchanges from baseline were greater in the high dose IC and IV groups ofpatients than in the low and mid dose IC groups. The persistence ofincreased ETT at 6 months (133.1 sec and 87.5 sec) in the high dose IC(24-48 μg/kg) and IV (18 & 38 μg/kg) groups, respectively, wasunexpected. The greatest mean increases in ETT of 107.9 and 133.1seconds at 2 and 6 months, respectively, occurred in the high dose(24-48 μg/kg) IC group. The IV group exhibited significant meanincreases in ETT of 93.4 seconds and 87.5 seconds, at 2 months and 6months respectively, which was not predicted by the rat and pig animalmodels used herein. Overall, the persistence of the effect (increase inETT) at six months and its magnitude for both the IC and IV groups waswholly unexpected.

The 66 human patients of the Phase I clinical trial described inExamples 1-3 herein were also evaluated using the Seattle AnginaQuestionnaire (SAQ). The SAQ is a validated, disease-specific, qualityof life instrument which assesses the following five scales: 1)“exertional capacity”=limitation of physical activity; 2) “diseaseperception”=worry about MI; 3) “treatment satisfaction”; 4) “anginafrequency”=number of episodes and sublingual nitroglycerin usage; and 5)“angina stability”=number of episodes with most strenuous physicalactivity. The possible range of scores for each of the five scales is 0to 100 with the higher scores indicating a better quality of life.Typically, a mean change of 8 points or more between the mean baselinescores (i.e., before treatment) and the post-treatment scores isrecognized as being “clinically significant.” However, in the presentanalysis, a dose was considered “effective” if the mean change in scorefrom baseline increased by greater than 14 points. The reason that 14was chosen (instead of 8) was to allow for the improvement that was seenin the placebo group at 2 months in a clinical trial of another growthfactor—VEGF.

In performing the SAQ evaluation, the patients were categorizedaccording to the same dosage groups that were evaluated for ETT, i.e.,0.33-2.0 μg/kg IC (low) 6.0-12.0 μg/kg IC (mid); 24-48 μg/kg IC (high);and 18 and 36 μg/kg IV. The questionnaire was administered to subjectsin each dosage group at baseline (prior to dosing), and at 2 months and6 months after being administered a single unit dose composition ofrFGF-2 in accordance with the method of the present invention.

The first SAQ scale is “exertional capacity.” The data on exertionalcapacity is summarized in Table 2 herein.

TABLE 2 Exertional Capacity (EC) - Change from Baseline FGF-2 Changefrom Baseline Change from Baseline Dose Group at Two Months at SixMonths 0.33 to 2.0 μg/kg IC N = 14 N = 7  (N = 16) 15.0* (−25 to 53)23.2* (0 to 53)  6.0 and 12 μg/kg IC N = 7  N = 6  (N = 8) 20.2* (−14 to44)  24.1 (−11 to 69) 24.0 to 48.0 μg/kg IC N = 26 N = 23 (N = 28) 14.6*(−33 to 75) 22.9* (−14 to 75) 18.0 and 36.0 μg/kg IV N = 12 N = 14 (N =14) 10.9 (−8 to 67) 16.5* (−19 to 63) N = number of subjects; mean(range); *= p < 0.05

As reflected in Table 2, the change from baseline in mean scoreincreased at 2 and 6 months for each of the three IC dosage groups andat 6 months for all dosage groups (IC and IV). All scores at all dosagelevels increased with time in going from 2 months to 6 months with thebest increases (23.2, 24.1, 22.9 and 16.5) relative to baseline beingseen at 6 months post-dosing.

The second SAQ scale to be evaluated was “angina stability.” The datasummarizing the angina stability is presented in Table 3 herein.

TABLE 3 Angina Stability (AS) - Change from Baseline FGF-2 Change fromBaseline Change from Baseline Dose Group at Two Months at Six Months0.33 to 2.0 μg/kg IC N = 13 N = 7 (N = 16) 46.2* (0 to 100) 21.4* (0 to50) 6.0 and 12 μg/kg IC N = 7  N = 6 (N = 8) 32.1* (0 to 50)    16.7(−25 to 50) 24.0 to 48.0 μg/kg IC N = 27 N = 24 (N = 28)  34.3* (−25 to75)  17.7* (−25 to 75) 18.0 and 36.0 μg/kg IV N = 12 N = 14 (N = 14)39.6* (0 to 100) 23.2* (0 to 75) N = number of subjects; mean (range);*= p < 0.05

According to Table 3, the change in score for angina stability increasedrelative to baseline at both 2 and 6 months for each group. Theimprovements in angina stability seen at 2 months after dosing (46.2,32.1, 34.3 and 39.6) were significantly greater than those scores seenat 6 months (21.4, 16.7, 17.7 and 23.2). However, the scores found atboth 2 months and 6 months after dosing showed that all dosages werefound to be effective (>14) in increasing angina stability. Moreover,the magnitude of the increases and their duration for 6 months wereunexpected.

The third SAQ scale to be evaluated was “angina frequency.” The datasummarizing the angina frequency is presented in Table 4 herein.

TABLE 4 Angina Frequency (AF) - Change from Baseline FGF-2 Change fromBaseline Change from Baseline Dose Group at Two Months at Six Months0.33 to 2.0 μg/kg IC N = 14 N = 7  (N = 16)  27.9* (−10 to 80) 12.9 (−40to 50) (low) 6.0 and 12 μg/kg IC N = 7 N = 6  (N = 8) 32.9* (0 to 80)36.7 (−10 to 90) (mid) 24.0 to 48.0 μg/kg IC N = 27 N = 24 (N = 28) 28.9* (−40 to 80) 25.8* (−30 to 80)  (high) 18.0 and 36.0 μg/kg IV N =12 N = 14 (N = 14) 20.0* (0 to 90) 11.4 (−30 to 60) ALL GROUPS N = 60 N= 51 (N = 66) 27.3 21.4 N = number of subjects; mean (range); *= p <0.05

According to Table 4, the mean patient scores (27.9, 32.9, 28.9 and20.0) for angina frequency increased at 2 months (relative to baseline)by an effective amount (>14) for all dosage groups and for all modes ofadministration (IC or IV). The mean patient scores continued to increaseat 6 months only for the mid dose (6.0-12.0 μg/kg) group, suggesting apeak effect at 2 months post-dosing. However, for the mid dose (6.0-12.0μg/kg) and high dose (24.0-48.0 μg/kg) groups, the changes at 2 monthsand 6 months were similar, suggesting a persistent effect at 6 months onangina frequency. The third SAQ scale to be evaluated was “anginafrequency.” The data summarizing the angina frequency is presented inTable 4 herein.

The fourth SAQ scale to be evaluated was “angina frequency.” The datasummarizing the angina frequency is presented in Table 5 herein.

TABLE 5 Treatment Satisfaction (TS) - Change from Baseline FGF-2 Changefrom Baseline Change from Baseline Dose Group at Two Months at SixMonths 0.33 to 2.0 μg/kg IC N = 14 N = 7  (N = 16) 8.5* (−19 to 31)  6.3(−25 to 25) (low) 6.0 and 12 μg/kg IC N = 7  N = 6  (N = 8) 17.9 (−13 to44) 19.8 (0 to 63)  (mid) 24.0 to 48.0 μg/kg IC N = 27 N = 24 (N = 28)18.8* (−38 to 69)   13.0 (−75 to 63) (high) 18.0 and 36.0 μg/kg IV N =12 N = 14 (N = 14) 19.8* (−13 to 63)  13.4* (−19 to 56) N = number ofsubjects; mean (range); *= p < 0.05

According to Table 5, the score for treatment satisfaction increased byan effective amount at 2 months for the mid and high dose IC groups aswell as the IV group. At six months post-dosing, only the score for themid dose group IC had a score that was greater than 14, suggesting apeak effect for treatment satisfaction at 2 months.

The fifth SAQ scale to be evaluated was “disease perception.” The datasummarizing the disease perception is presented in Table 6 herein.According to Table 6, the scores for disease perception increased frombaseline to scores of 20.2-29.2 at 2 months and 23.8-34.0 at 6 months.These scores showed that administering a single unit dose composition inaccordance with the method of the present invention was considered to beas effective (or more effective) at 6 months as at two months. Thesescores suggest a persistence of the effectiveness of the method of thepresent invention on disease perception out to six months followingadministration of a single unit dose composition.

TABLE 6 Disease Perception (DP) - Change from Baseline Change fromBaseline Change from Baseline Dose Group at Two Months at Six Months0.33 to 2.0 μg/kg IC N = 14 N = 7  (N = 16) 29.2* (−8 to 58) 26.2* (0 to42)  (low) 6.0 and 12 μg/kg IC N = 7  N = 6  (N = 8) 20.2* (−8 to 50)30.6* (0 to 58)  (mid) 24.0 to 48.0 μg/kg IC N = 27 N = 24 (N = 28) 27.8* (−33 to 92)  34.0* (−33 to 100) (high) 18.0 and 36.0 μg/kg IV N =12 N = 14 (N = 14) 22.9* (−8 to 92) 23.8* (−8 to 75) N = number ofsubjects; mean (range); *= p < 0.05

Up to 60 of the human patients of the Phase I clinical trial describedin Examples 1-3 herein were also evaluated using resting magneticresonance imaging (MRI) scans of their heart. The resting MRI scans wereperformed on the patients at baseline, and at 1 month, 2 months and 6months after dosing with a single unit dose composition of the presentinvention. The doses were considered “effective” based upon statisticalsignificance (p<0.05). The objective criteria assessed by the restingMRI scans are the following: (1) ejection fraction; (2) myocardialinfarct extent (%); (3) normal wall thickening (4) normal wall motion(%); (5) target wall thickening (%); (6) target wall motion (%); (7)target wall area collateral extent (%); and (8) target area delayedarrival extent (%).

Based upon the resting MRI, no change in “ejection fraction” wasobserved at one month for any one group. The mean change from baselinefor all groups (n=33) at 1 month was an increase of 2.0% (p=0.042). Attwo months, the mean change from baseline for the low dose IC group(n=13) was an increase of 8.1% (p=0.007); and for all groups (n=54), themean change from baseline was an increase of 3.8% (p=0.001). At sixmonths, the mean change from baseline for the high dose IC group (n=19)was 5.3% (p=0.023); for the IV group (n=3) was 11.1% (p=0.087); and forall groups (n=33) was 5.7% (p=0.001).

Based upon the resting MRI, there was no statistically significantchange in the “myocardial infarct extent” (%) for any group, or for allgroups in combination at 1 month, 2 months or 6 months post-dosing. Whenthe normal wall motion (%) and normal wall thickening were assessed,there was no statistically significant change from baseline at 1 month,2 months or 6 months for any one group. However, there was astatistically significant change from baseline in target wall motion forall groups at one (n=60), two (n=54) and six (n=33) months, which wasreflected as a mean increase from baseline of 2.7% (p=0.015), 4.4%(p=<0.001) and 6.4% (p<0.001), respectively. However, there was also astatistically significant change from baseline in target wall thickeningfor all groups at one (n=60), two (n=54) and six (n=33) months, whichwas reflected as a mean increase from baseline of 4.4% (p=0.015), 6.3%(p=<0.001) and 7.7% (p<0.001), respectively.

The next criteria assessed by MRI was “target area collateral extent”(%). The mean increase from baseline in target area collateral extentfor all groups was highly statistically significant at one month (n=31),two months (n=27) and six months (n=16), wherein the increases were 8.3%(p<0.001), 10.9% (p<0.001) and 11.2% (p<0.001), respectively. Thegreatest collateral extent increases were observed for the low and midIC doses, i.e., at one month (10.4% and 18.3%, respectively), two months(14.7% and 18.0%, respectively) and six months (16.0% and no value formid dose, respectively), which was wholly unexpected. At one month, twomonths and six months post-dosing, the corresponding % increases intarget area collateral extent that were observed for the IC high dosegroup were 6.3%, 8.0% and 9.0%, respectively.

The final criteria assessed by MRI was “target area delayed arrivalextent” (%). The mean decrease from baseline in target area delayedarrival extent for all groups was highly statistically significant at 1month (n=60), 2 months (n=54) and 6 months (n=34), wherein the decreaseswere −5.8% (p<0.001), −8.3% (p<0.001) and −10.0% (p<0.001),respectively. The greatest target area delayed extent decreases wereobserved for the low dose IC group, which was also highly unexpected.

Thus, providing CAD patients with a single IC or IV infusion of rFGF-2in accordance with the present invention provided the patients with astatistically significant physical improvement as objectively measuredby MRI and other conventional criteria.

Pharmacokinetics and Metabolism

The molecular structure of FGF-2 contains a positively charged tail thatis known to bind to proteoglycan chains (heparin and heparin-likestructures) on cell surfaces and on the endothelial wall of thevasculature. See Moscatelli, et al., “Interaction of Basic FibroblastGrowth Factor with Extracellular Matrix and Receptors,” Ann. NY Acad.Sci., 638:177-181 (1981).

The kidneys and liver are the major organs for the elimination ofrFGF-2. In particular, the kidneys have a protein cutoff of about 60 kDand thus retain serum albumin (MW 60 kD). However, FGF-2 (146 residues)has a molecular weight of about 16.5 kD. Accordingly, renal excretion isto be expected. In a radiolabelled biodistribution study of commerciallyavailable bovine FGF-2 (bFGF-2), both the liver and the kidney wereshown to contain high counts of the radiolabelled bFGF-2 at 1 hour afterIV or IC injection. In a published study, wherein another recombinantiodinated form of bFGF-2 was given to rats, the liver was identified asthe major organ of elimination. Whalen et al., “The Fate ofIntravenously Administered bFGF and the Effect of Heparin,” GrowthFactors, 1:157-164 (1989). It is also known that FGF-2 binds in thegeneral circulation to α₂-macroglobulin and that this complex isinternalized by receptors on the Kupffer cells. Whalen et al. (1989) andLaMarre et al., “Cytokine Binding and Clearance Properties ofProteinase-Activated Alpha-2-Macroglobulins,” Lab. Invest., 65:3-14(1991). Labelled FGF-2 fragments were not found in the plasma, but theywere found in the urine and corresponded in size to intracellularbreakdown products.

In preclinical testing, we determined the pharmacokinetics of rFGF-2(SEQ ID NO:2) after intravenous (IV) and intracoronary (IC)administration in domestic Yorkshire pigs, and after IV administrationdosing in Sprague Dawley (“SD”) rats. The pig models demonstrated linearpharmacokinetics (0.65 μg/kg-20 μg/kg) IC and IV. The terminal half-lifeof the FGF-2 in the pig model was 3-4 hours. The rat models demonstratedlinear pharmacokinetics over the range of 30-300 μg/kg IV. The terminalhalf-life of the FGF-2 in the rat model was 1 hour. Both species showedplasma concentration suggesting a two-compartment model.

Likewise, in humans, the FGF-2 plasma concentrations after IV and/or ICinfusion followed a biexponential curve with an initial steep slope andconsiderable decrease over several log scales (the distribution phase)during the first hour, followed by a more moderate decline (theelimination phase). FIG. 1A provides a plasma concentration versus timecurve showing these phases in humans after IC administration of rFGF-2of SEQ ID NO:2 as a function of each of the following eight doses: 0.33μg/kg, 0.65 μg/kg, 2 μg/kg, 6 μg/kg, 12 μg/kg, 24 μg/kg, 36 μg/kg, and48 μg/kg of lean body mass (LBM). FIG. 1A shows the plasma doselinearity for the eight doses of rFGF-2 that were administered by ICinfusion over a twenty minute period. FIG. 1A also shows a biphasicplasma level decline, i.e., a fast distribution phase during the firsthour, followed by an elimination phase with an estimated T_(1/2) of 5-7hours. The plasma concentrations of FGF-2 of SEQ ID NO:2 were determinedby a commercially available ELISA (R&D Systems, Minneapolis Minn.) thatwas marketed for analysis of human FGF-2. The ELISA assay showed 100%cross-reactivity with the rFGF-2 of SEQ ID NO:2. Other members of theFGF family, as well as many other cytokines, were not detected by thisassay. Further, heparin does not interfere with the assay.

FIG. 1B is a plot of the mean FGF-2 plasma concentration as a functionof time for 18 μg/kg and 36 μg/kg rFGF-2 administered IV, as compared to36 μg/kg rFGF-2 administered IC. The plasma concentration versus timeprofiles in FIG. 1B for the 36 μg/kg doses by the IV and IC routes aresuperimposible. However, a first-pass effect with the IC route is noteliminated.

FIG. 2 is a plot of mean FGF-2 area under the curve (AUC) in pg*min/mlcorresponding to FIGS. 1A and 1B. FIG. 2 shows the dose linearity ofsystemic rFGF-2 exposure (AUC) following IC or IV infusion. Inparticular, FIG. 2 shows that the systemic exposure to rFGF-2 followingadministration by the IC and IV routes is substantially similar.

FIG. 3 is a plot of individual human patient plasma clearance (CL)values (ml/min/kg) as a function of the time of heparin administrationin “minutes prior to rFGF-2 infusion.” FIG. 3 shows the influence oftiming of heparin administration on FGF-2 plasma clearance (CL).Although FIG. 3 shows that administering heparin up to 100 minutes priorto rFGF-2 decreases FGF-2 clearance, the preferred time foradministering heparin is from 0-30 minutes prior the rFGF-2administration, wherein the effect of the heparin on decreasing FGF-2clearance is greatest.

FIG. 4 is a plot OF individual human patient rFGF-2 dose normalized areaunder curves (AUCs) as a function of the time of heparin administrationin “minutes prior to rFGF-2 infusion” and shows the influence of timingof heparin administration on rFGF-2 AUC. FIG. 4 shows that the greatestAUC/dose was achieved when an effective amount of a glycosoaminoglycan,such as heparin, was preadministered within 30 minutes or less of ICrFGF-2 infusion, more preferably within 20 minutes or less of IC or IVrFGF-2 infusion. Typically, an effective amount of a glycosoaminoglycanis 10-80 U/kg heparin.

The mean pharmacokinetic parameters for rFGF-2 in humans as a functionof dosage and mode of administration are summarized in Table 8 herein.Referring to Table 8, the T½ for FGF-2 in humans was determined to rangefrom 2.2±3.7 hours at low dose (0.33-2.0 μg/kg) IC to 7.0±3.5 hours at adose of 18-36 μg/kg IV; given the limitations of the assay, the terminalhalf-life is estimated at 5-7 hours for all groups. The clearances ofFGF-2 ranged from 13.2 to 18.2 L/hour/70 kg man. Finally, the steadystate volume (V_(ss)) was determined to range from 11.3±10.4 L/70 kg manto 16.8±10.7 L/70 kg man.

TABLE 8 Mean rFGF-2 PK Parameters in Humans FGF-2 CL V_(ss) Dose μg/kg NRoute (L/hr/70 kg) t_(1/2) (h) (L/70 kg) 0.3-2   16 IC  18.2 ± 13.4 2.2± 3.7 11.3 ± 10.4  6-12 8 IC 13.2 ± 7.3 3.1 ± 2.5 12.1 ± 4.9  24-48 28IC 14.7 ± 8.3 6.3 ± 1.8 16.8 ± 10.7 18-36 14 IV 13.9 ± 7.9 7.0 ± 3.516.4 ± 8.6 

Although the binding of FGF-2 to heparin-like structures is strong(dissociation constant ˜2×10⁻⁹ M), the binding of FGF-2 to a specifictyrosine kinase receptor is approximately two orders of magnitude higher(dissociation constant ˜2×10⁻¹¹ M). Moscatelli et al., (1991). Thus,without being bound to any theory, the complexation of rFGF-2 with aglycosoaminoglycan, such as a heparin, might increase signaltransduction and mitogenesis, and/or protect the rFGF-2 from enzymaticdegradation.

The examples, which follow, provide more details on the selectioncriterion and the Phase I clinical trial that gave rise to the datadiscussed above.

Example 1 Unit Dose of rFGF-2 Employed in the Phase I Clinical Trial

The rFGF-2 of SEQ ID NO:2 was formulated as a unit dose andpharmaceutical composition and administered to rats, pigs and ultimatelyto humans in the Phase I clinical trial referenced herein. The variousformulations are described below.

The rFGF-2 unit dose was provided as a liquid in 3 cc type I glass vialswith a laminated gray butyl rubber stopper and red flip-off overseal.The rFGF-2 unit dose contained 1.2 ml of 0.3 mg/ml rFGF-2 of SEQ ID NO:2in 10 mM sodium citrate, 10 mM monothioglycerol, 1 mM disodium dihydrateEDTA (molecular weight 372.2), 135 mM sodium chloride, pH 5.0. Thus, inabsolute terms, each vial (and unit dose) contained 0.36 mg rFGF-2. Thevials containing the unit dose in liquid form were stored at 2° to 8° C.

The rFGF diluent was supplied in 5 cc type I glass vials with alaminated gray butyl rubber stopper and red flip-off overseal. TherFGF-2 diluent contains 10 mM sodium citrate, 10 mM monothioglycerol,135 mM sodium chloride, pH 5.0. Each vial contained 5.2 ml of rFGF-2diluent solution that was stored at 2° to 8° C.

The rFGF-2 pharmaceutical composition that was infused was prepared bydiluting the rFGF-2 unit dose with the rFGF diluent such that theinfusion volume is 10-40 ml. In order to keep the EDTA concentrationbelow a preset limit of 100 μg/ml, the total infusion volume wasincreased to a maximum of 40 ml when proportionately higher absoluteamounts of FGF-2 were administered to patients with higher body weights.

Example 2 Selection Criteria For Patients With Coronary Artery DiseaseFor Treatment With rFGF-2

The following selection criteria were applied to Phase I patients withcoronary artery disease, whose activities were limited by coronaryischemia despite optimal medical management, and who were not candidatesfor approved revascularization therapies:

Inclusion Criteria:

Subject is eligible if:

-   -   Male or female, greater than or equal to 18 years of age    -   Diagnosis of coronary artery disease (CAD)    -   Suboptimal candidates for approved revascularization procedures,        e.g., angioplasty, stents, coronary artery bypass graft (CABG)        (or refuses those interventions)    -   Able to exercise at least three minutes using a modified Bruce        protocol and limited by coronary ischemia    -   Inducible and reversible defect of at least 20% myocardium on        pharmacologically stressed thallium sestamibi scan    -   CBC, platelets, serum chemistry within clinically acceptable        range for required cardiac catheterization    -   Normal INR, or if anticoagulated with Coumadin, INR <2.0    -   Willing and able to give written informed consent to participate        in this study, including all required study procedures and        follow-up visits

Exclusion Criteria:

Subject is not eligible if:

-   -   Malignancy: any history of malignancy within past ten years,        with the exception of curatively treated basal cell carcinoma    -   Ocular conditions: proliferative retinopathy, severe        non-proliferative retinopathy, retinal vein occlusion, Eales'        disease, or macular edema or funduscopy by ophthalmologist:        history of intraocular surgery within six months    -   Renal function: creatinine clearance below normal range adjusted        for age; protein >250 mg or microalbumin >30 mg/24 h urine    -   Class IV heart failure (New York Heart Association)    -   Ejection fraction <20% by echocardiogram, thallium scan, MRI or        gated pooled blood scan (MUGA)    -   Hemodynamically relevant arrhythmias (e.g., ventricular        fibrillation, sustained ventricular tachycardia)    -   Severe valvular stenosis (aortic area <1.0 cm², mitral area <1.2        cm²), or severe valvular insufficiency    -   Marked increase in angina or unstable angina within three weeks    -   History of myocardial infarction (MI) within three months    -   History of transient ischemic attack (TIA) or stroke within six        months    -   History of CABG, angioplasty or stent within six months    -   History of treatment with transmyocardial laser        revascularization, rFGF-2, or vascular enodothelial growth        factor (VEGF) within six months    -   Females of child-bearing potential or nursing mothers    -   Any pathological fibrosis, e.g., pulmonary fibrosis, scleroderma    -   Known vascular malformation, e.g., AV malformation, hemangiomas    -   Coexistence of any disease which might interfere with assessment        of symptoms of CAD, e.g., pericarditis, costochondritis,        esophagitis, systemic vasculitis, sickle cell disease    -   Coexistence of any disease which limits performance of modified        Bruce protocol exercise stress test, e.g., paralysis or        amputation of a lower extremity, severe arthritis or lower        extremities, severe chronic obstructive pulmonary disease (COPD)    -   Participation in clinical trials of investigational agents,        devices or procedures within thirty days (or scheduled within        sixty days of study drug)    -   Known hypersensitivity to rFGF-2 or related compounds    -   Any condition which makes the subject unsuitable for        participation in this study in the opinion of the investigator,        e.g., psychosis, severe mental retardation, inability to        communicate with study personnel, drug or alcohol abuse.

Example 3 Phase I Clinical Study on Recombinant FGF-2 (SEQ ID NO:2)Administered to Humans

The Phase I CAD trial of this example is an open label, dose escalationstudy of recombinant fibroblast growth factor-2 (rFGF-2) for safety,tolerability and pharmacokinetics. The study was conducted at two sites:Beth Israel Deaconess Hospital (Harvard) in Boston, Mass. and EmoryUniversity Hospital in Atlanta, Ga. Enrollment is complete. Subjectswere treated with a single infusion of rFGF-2 on Day 1 and followed for360 days; follow-up is not yet complete in some subjects.

The subject population consists of patients with advanced CAD who areexercise limited by coronary ischemia and are considered suboptimalcandidates for (or do not want to undergo) one of the establishedrevascularization procedures (e.g., CABG, angioplasty—with or withoutstent). The major exclusion criteria were history or suspicion ofmalignancy, uncompensated heart failure or left ventricular ejectionfraction <20%, renal insufficiency or proteinuria, and various ocularconditions (e.g., proliferative diabetic retinopathy, severenon-proliferative retinopathy).

Sixty-six subjects have received rFGF-2 of SEQ ID NO:2 in this trial:fifty-two received the rFGF-2 as an IC infusion and fourteen received itas an IV infusion. Each subject was observed in the hospital for atleast twenty-four hours, and then followed as an outpatient for 360 dayswith follow-up visits (Days 15, 29, 57, 180 and 360). At least foursubjects were studied at each dose; if no subject experienceddose-limiting toxicity as defined by the protocol within six days, thedose was escalated. The drug was administered as a single 20 minuteinfusion divided between two major sources of coronary blood supply(IC), using standard techniques for positioning a catheter into thepatient's coronary artery (such as already employed in angioplasty) orin a peripheral vein (IV). The doses in μg/kg of rFGF-2 administered IC(and the number of patients) were 0.33 (n=4), 0.65 (n=4), 2.0 (n=8), 6.0(n=4), 12.0 (n=4), 24 (n=8), 36 (n=10) and 48 (n=10) of rFGF-2 of SEQ IDNO:2. The doses in μg/kg of rFGF-2 administered IV (and the number ofpatients) were 18.0 (n=4) and 36.0 (n=10) or rFGF-2 of SEQ ID NO: 2.

Angina frequency and quality of life was assessed by the Seattle AnginaQuestionnaire (SAQ) at a baseline (before rFGF-2 administration) and at2 months and 6 months after rFGF-2 administration. Exercise tolerancetime (ETT) was assessed by the treadmill test at 1, 2, and 6 months.Rest/exercise nuclear perfusion and gated sestamibi-determined restejection fraction (EF), and resting magnetic resonance imaging (MRI)were assessed at baseline, and at 1 month, 2 months and 6 months postrFGF-2 administration. MRI measurements which were thought toobjectively measure changes in % in cardiac function and perfusionincluded: (1) ejection fraction; (2) myocardial infarct extent (%); (3)normal wall thickening (4) normal motion (%); (5) target wall thickening(%); (6) target wall motion (%); (7) target wall area collateral extent(%); and (8) target area delayed arrival extent (%).

If one of four subjects experienced dose-limiting toxicity (as definedby the protocol), four additional subjects were studied at that dose; ifnone experienced toxicity, the dose was escalated and another group wasstudied. The maximally tolerated dose (MTD) was defined as the IC dosewhich was tolerated by 9/10 subjects, i.e., 36 μg/kg IC.

Careful fluid management pre-infusion was prescribed using a Swan-Ganzcatheter and vital signs were monitored frequently during dosing.Heparin was administered IV prior to the infusion of rFGF-2 in allgroups. The EDTA concentration was less than 100 μg/ml in the unit dosecomposition. Volume of study drug administered varied with dose andsubject's weight, and ranged from 10 ml at lower doses to 40 ml athigher doses.

Preliminary Results

The results presented here are unaudited and are based on a thirdinterim analysis for sixty-six subjects with six months follow-up forall groups (1-10) and the serious adverse events (SAE) report of 29 Jul.1999 from Chiron Drug Safety. Data collection for the last visit (Day360) and final analysis is in progress.

The starting dose of 0.33 μg/kg IC was escalated over eight sequentialgroups to 48 μg/kg IC, at which dose 2 of ten subjects experienceddose-limiting toxicity (hypotension) as defined by the protocol.Hypotension was manageable with fluids alone in all subjects (novasopressors or assistive devices). At 36 μg/kg IC, only 1 of 10subjects had dose-limiting toxicity which defined this dose as themaximally tolerated dose (MTD). Two additional groups were studied by IVinfusion; four subjects of half the MTD (18 μg/kg) and ten subjects atthe MTD (36 μg/kg).

Hypotension was dose-limiting in humans, as predicted by the animalmodel in Yorkshire pigs. However, 36.0 μg/kg rFGF-2 IC was tolerated inhumans; whereas in pigs, 20.0 μg/kg rFGF-2 IC caused profoundhypotension in one of two animals. Better tolerability in humans wasattributed to aggressive fluid management and absence of generalanesthesia.

As of 29 Jul. 1999, thirty-three serious adverse events (SAEs) haveoccurred in 24/66 subjects, but were not dose-related. Fifteen (15) SAEswere considered at least possibly related to rFGF-2; whenever there wasa difference between relatedness assigned by the investigator and themedical monitor, the more conservative relationship was assigned. SAE'swere multiple in five subjects: 01103 (0.33 μg/kg IC), 01106 (0.65 μg/kgIC), 01113 (2.0 μg/kg IC), 01137 (36.0 μg/kg IV), 02101 (0.65 μg/kg IC).

The most frequent treatment-emergent adverse events (AEs) on Day 1 weretransient systolic hypotension and transient bradycardia. Thehypotension was dose-dependent and occurred more frequently at dosesgreater than or equal to (≧) 24 μg/kg IC; bradycardia was notdose-dependent. Other adverse events (AEs) which were deemed at leastpossibly related and appeared dose-related occurred within the firstseveral days or week post infusion and included chest pain, shortness ofbreath, insomnia, anxiety, and nausea. These events were mild tomoderate in severity, and most did not require specific intervention.

When administered IC, the drug was administered over approximately 20minutes as a single infusion divided between two major sources ofcoronary blood supply (IC), using standard techniques for positioning acatheter into the patient's coronary artery (such as already employed inangioplasty). When administered IV, the drug was administered as aninfusion over 20 minutes into a peripheral vein.

The preliminary safety results indicate that serious events were notdose related. Thus far, of the eight IC dosage groups, there were threedeaths in the lower dosage groups, i.e., at 0.65 μg/kg (Day 23), at 2.0μg/kg (Day 57) and at 6.0 μg/kg (Day 63), and one death in the highestdose group, i.e., at 48.0 μg/kg (approximately 4 months post-dosing).Three of the deaths were cardiac; one death was due to a large B celllymphoma that was diagnosed three weeks after dosing in the patient ingroup 4 (6.0 μg/kg) who patient died at two months post-dosing.

Acute myocardial infarction (MI) occurred in four patients, i.e., onepatient from each of groups 1 (0.33 μg/kg), 3 (2.0 μg/kg), 4 (6.0 μg/kg)and 7 (36.0 μg/kg). Multiple MIs occurred in two patients, i.e., onefrom group 1 (0.33 μg/kg) and one from group 3 (2.0 μg/kg). Emergencyrevascularization procedures (CABG or angioplasty with or without stent)were performed during follow-up in 4 patients: one each from groups 1(0.33 μg/kg), 3 (2.0 μg/kg), 4 (6.0 μg/kg), and 7 (36.0 μg/kg).

Acute hypotension, seen at higher doses during or just subsequent toinfusion, was managed by administration of IV fluids without need for avasopressor. The maximally tolerated dose (MTD) in humans was defined as36 μg/kg IC. (In contrast, in pigs, the MTD was 6.5 μg/kg IC.) Doses ofrFGF-2 up to 48 μg/kg IC were administered in human patients withaggressive fluid management, but were defined by the protocol as “nottolerated” due to acute and/or orthostatic hypotension in two out of tenpatients. The terminal half-life of the infused rFGF-2 was estimated at5 to 7 hours.

The human patients in this study that were treated with a single IC orIV infusion of rFGF-2 of SEQ ID NO:2 exhibited a mean increase in ETT of1.5 to 2 minutes. See Table 1. This is especially significant because anincrease in ETT of greater than (>) 30 seconds is considered significantand a benchmark for evaluating alternative therapies, such asangioplasty. The angina frequency and quality of life, as measured bySAQ, showed a significant improvement at 2 months in all five subscalesfor the 66 patients (n=66) tested. See Tables 26. In Tables 2-6, a meanchange of 14 or more was considered “clinically significant.”

When 33 human CAD patients were assessed by resting cardiac magneticresonance imaging (MRI) at baseline, and at 1, 2, and 6 months afterreceiving a single unit dose composition of the present invention by ICor IV routes, a highly statistically significant increase was observedin target wall thickening, target wall motion and target area collateralextent; a highly statistically significant decrease was observed intarget area delayed arrival extent; and no statistically significantchanges were observed in normal wall motion, normal wall thickening ormyocardial infarct extent.

In addition to the above criterion (i.e., ETT, SAQ, MRI), a treatment isconsidered very successful if the angiogenic effects last at least sixmonths. In the present Phase I study, the unexpectedly superiorangiogenic effects were observed to last up to 6 months in some patientsin all dosage groups. Based upon the results already obtained, it isexpected that the angiogenic effects may last twelve months or more butdo last at least six months in the patients, at which time the procedurecould be repeated, if necessary.

Example 4 Proposed Phase II Clinical Study On rFGF-2 (SEQ ID NO:2)Administered to Humans to Treat Coronary Artery Disease

The Phase II clinical trial of rFGF-2 for treating human patients forcoronary artery disease is performed as a double blind/placebocontrolled study having four arms: placebo, 0.3 μg/kg, 3.0 μg/kg, and 30μg/kg administered once IC. This study is ongoing and results are notyet available.

Example 5 Unit Dose and Pharmaceutical Composition of rFGF-2 for thePhase II Human Clinical Trial

The rFGF-2 of SEQ ID NO:2 was formulated as a unit dose pharmaceuticalcomposition for administration to humans in the Phase II clinical trialreferenced herein. The various formulations are described below.

The rFGF-2 unit dose was prepared as a liquid in 5 cc type I glass vialswith a laminated gray butyl rubber stopper and red flip-off overseal.The rFGF-2 formulation contains 0.3 mg/ml rFGF-2 of SEQ ID NO:2 in 10 mMsodium citrate, 10 mM monothioglycerol, 0.3 mM disodium dihydrate EDTA(molecular weight 372.2), 135 mM sodium chloride, pH 5.0. Each vialcontained 3.7 ml of rFGF-2 drug product solution (1.11 mg rFGF-2 pervial). The resulting unit dose in liquid form is stored at less than−60° C. The above described unit dose is diluted with the “rFGF-2placebo.”Depending on the size of the patient, the contents of severalof the vials may be combined to produce a unit dose of 36 μg/kg for thePhase II study.

The rFGF-2 placebo is supplied as a clear colorless liquid in 5 cc typeI glass vials with a laminated gray butyl rubber stopper and redflip-off overseal. The rFGF-2 placebo is indistinguishable in appearancefrom the drug product and has the following formulation: 10 mM sodiumcitrate, 10 mM monothioglycerol, 0.3 mM disodium dihydrate EDTA(molecular weight 372.2), 135 mM sodium chloride, pH 5.0. Each vialcontains 5.2 ml of rFGF-2 placebo solution. Unlike the unit dose, therFGF-2 placebo is stored at 2° to 8° C.

The rFGF-2 pharmaceutical composition that is infused is prepared bydiluting the rFGF-2 unit dose with the rFGF diluent such that theinfusion volume is 20 ml for Phase II.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference in its entirety.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention described herein.

1. A method for treating a human patient for coronary artery disease,comprising administering a therapeutically effective amount of arecombinant fibroblast growth factor-2 (FGF-2) or an angiogenicallyactive fragment or an angiogenically active mutein thereof into one ormore coronary vessels or into a peripheral vein in a human patient inneed of treatment for said coronary artery disease, said therapeuticallyeffective amount being about 0.2 μg/kg to 48 μg/kg of patient weight,wherein said recombinant FGF-2 has the amino acid sequence of humanFGF-2, and wherein said angiogenically active mutein has at least 90%amino acid sequence identity with human FGF-2.
 2. The method of claim 1,further comprising the step of administering to said human patient about10 U/kg to 80 U/kg of heparin within 30 minutes of administering saidrecombinant FGF-2 or said angiogenically active fragment or saidangiogenically active mutein thereof.
 3. The method of claim 2, whereinsaid therapeutically effective amount of said recombinant FGF-2 or saidangiogenically active fragment or said angiogenically active muteinthereof is administered into one or more coronary vessels.
 4. The methodof claim 3, wherein said therapeutically effective amount of saidrecombinant FGF-2 or said angiogenically active fragment or saidangiogenically active mutein thereof is about 24 μg/kg to 48 μg/kg. 5.The method of claim 2 wherein said therapeutically effective amount ofsaid recombinant FGF-2 or said angiogenically active fragment or saidangiogenically active mutein thereof is administered into a peripheralvein.
 6. The method of claim 5, wherein said therapeutically effectiveamount of said recombinant FGF-2 or said angiogenically active fragmentor said angiogenically active mutein thereof is about 18 μg/kg to 36μg/kg.
 7. The method of claim 1, wherein said therapeutically effectiveamount of said recombinant FGF-2 or said angiogenically active fragmentor said angiogenically active mutein thereof is administered byinfusion.
 8. A method for treating a human patient for coronary arterydisease or a myocardial infarction, comprising administering a singleunit dose of a recombinant fibroblast growth factor-2 (FGF-2) or anangiogenically active fragment or an angiogenically active muteinthereof into one or more coronary vessels or into a peripheral vein in ahuman patient in need of treatment for coronary artery disease or amyocardial infarction, said unit dose comprising from about 0.008 mg to7.2 mg of said recombinant FGF-2 or said angiogenically active fragmentor said angiogenically active mutein thereof, wherein said recombinantFGF-2 has the amino acid sequence of human FGF-2, and wherein saidangiogenically active mutein has at least 90% amino acid sequenceidentity with human FGF-2.
 9. The method of claim 8, wherein said singleunit dose produces a therapeutic benefit against coronary artery diseasein said human patient that lasts at least four months.
 10. The method ofclaim 9, wherein said therapeutic benefit in said human patient lasts 6months.
 11. The method of claim 10, wherein said therapeutic benefit isof such magnitude and duration in said human patient such thatadministration of a second unit dose is not required for about 6 months.12. The method of claim 10, wherein said unit dose comprises 0.3 mg to3.5 mg of said recombinant FGF-2 or said angiogenically active fragmentor said angiogenically active mutein thereof.
 13. The method of claim 8,further comprising the step of administering 10 U/kg to 80 U/kg ofheparin to said patient within 30 minutes of administering said unitdose, wherein said heparin is administered by intravenous orintracoronary administration.
 14. The method of claim 8, wherein saidunit dose is administered by infusion.
 15. A method for inducingangiogenesis in a heart of a human patient, comprising administering asingle unit dose of a recombinant fibroblast growth factor-2 (FGF-2) oran angiogenically active fragment or an angiogenically active muteinthereof into one or more coronary vessels or into a peripheral vein in ahuman patient in need of treatment for coronary artery disease, saidunit dose comprising from about 0.008 mg to 7.2 mg of said recombinantfibroblast growth factor-2 (FGF-2) or said angiogenically activefragment or said angiogenically active mutein thereof, wherein saidrecombinant FGF-2 has the amino acid sequence of human FGF-2, andwherein said angiogenically active mutein has at least 90% amino acidsequence identity with human FGF-2.
 16. The method of claim 15, whereinsaid single unit dose produces an improvement in one or more clinicalendpoints in said human patient that lasts at least four months.
 17. Themethod of claim 16, wherein said single unit dose produces animprovement in one or more clinical endpoints in said human patient thatlasts 6 months.
 18. The method of claim 15, wherein said unit dose isadministered by infusion.
 19. A method for providing a human patientwith relief from symptoms of angina, comprising administering a singleunit dose of a recombinant fibroblast growth factor-2 (FGF-2) or anangiogenically active fragment or an angiogenically active muteinthereof into one or more coronary vessels or into a peripheral vein in ahuman patient in need of relief from symptoms of angina, said unit dosecomprising from about 0.008 mg to 7.2 mg of said recombinant fibroblastgrowth factor-2 (FGF-2) or said angiogenically active fragment or saidangiogenically active mutein thereof, wherein said recombinant FGF-2 hasthe amino acid sequence of human FGF-2, and wherein said angiogenicallyactive mutein has at least 90% amino acid sequence identity with humanFGF-2.
 20. The method of claim 19, wherein said unit dose isadministered by infusion.